| ObjectiveTo imProve and Perfect the trophoblastic cells primary culture method s,and identification of the cultured cells.Establish the trophoblastic cells damage model induced by mifepristone.To observe the effect of the active ingredients of the modified STW on the trophoblastic cells damage model,to bring out the optimal extraction method,the optimal proportion and the compatibility rule of the active ingredients of the modified STW.MethodsExperiment 1:The placental villus were digested by 0.25%tryPsin,puri fied by percoll density gradient method and differential adhesion method,and identificated by inverted microscope,electron microscopy,immunohis tochemistry,flow cytometry,the level of ?-hCG in cell supernatant.Experiment 2:The trophoblastic cells were induced by different conce ntrations of mifepristone for 24h and 48h?then retreat 24h?48h.The trop hoblastic cells proliferation was detected by MTT assay.The early stage apoptosis detected with AnnexinV FITC/PI by flow cytometry.The morpholog y of apoptosis is assessed by Hochest33258 dye and electron microscopy.T he Protein expression of Caspase-3,Bax,Bcl-2 detected with Western blot and the expression of Caspase-3,Bax,Bcl-2 mRNA detected with PCR induc ed by mifepristone for 24h and retreat 24h.Experiment 3:The five active ingredients of 10 concentration of the modified STW were divided in 10 groups by uniform design.The trophoblas tic cells proliferation induced by mifepristone was detected by MTT assay.The early stage apoptosis detected with AnnexinV FITC/PI by flow cytomet ry.The protein expression of Caspase-3 was detected with Western blot.The ?-HCG of trophoblastic cells supernatant was assessed by radioimmuno assay.The analysis of result was by means of stepwise regression analysi s,canonical correlation analysis and optimization results analysis.Experiment 4:The optimum combination was further verified by trophob lastic cells damaged model.The trophoblastic cells proliferation induced by mifepristone was detected by MTT assay.The early stage apoptosis det ected with Annexin V FITC/PI by flow cytometry.The protein expression of Caspase-3,Caspase-9,Bax,Bcl-2,Xiap was detected with Western blot.The expression of Caspase-3,Caspase-9,Bax,Bcl-2,Xiap mRNA detected wi th PCR.ResultsExPeriment 1:Under the electron microscope,there were abundance mic rovillus and alveolus in cell surface,the cytoplasm is rich with obvious lipid drops,significantly nuclear compared to kytoplasma.The positive expression of cytokeratin and weakly positive expression of vimentin were found by immunohistochemical and flow cytometry.Cell supernatants ?-hC G level was 913.27MIU/ml primary cultured.after 24h,then decreased gradu ally.Experiment 2:Induced in 24h,There was no significant difference be tween groups on the proliferation of trophoblastic(P>0.05).There was n o significant difference between groups on the Proliferation of trophobla stic(P>0.05).The early apoptosis rate of 20 ? mol/L,40 ? mol/L,60?mo 1/L,80? mol/L,100? mol/L group was significantly increased compared to the blank control group(P<0.01);The Caspase-3 protein in 100?mol/L gr oup was increased than the blank control group(P<0.05);The Bax protei n expression was significantly decreased of 80 ? mol/L,100?mol/L group c ompared to the blank control group(P<0.01)and in group 40? mol/L?60? m ol/L was decreased to the blank control group(P<0.05);The expression o f Bcl-2 in group 100?mol/L and 80?mol/L was significantly increased com pared to the blank control group,in group 60? mol/L was increased compar ed to the blank control group(P<0.01).The bcl-2/Bax in 60? mol/L group was decreased to the blank control group(P<0.05),in 80?mol/L,100 ? mol/L group was significantly decreased to the blank control group(P<0.01);Retreated in 24h,the trophoblastic cell proliferation in 40 mu mol/L,60 mu mol/L,80 ?mol/L,100 mu mol/L group was significantly decreas ed than the control blank group(P<0.01);The early apoptosis rate in gr oup 60? mol/L,801? mol/L,100 mu mol/L was increased significantly than t he blank control group(P<0.01);The Caspase-3 Protein in 80? mol/L and 60? mol/group was increased to the 20? mol/L group(P<0.05);The Bax protein expression was decreased in 80? mol/L,60? mol/L group compared to the blank control group(P<0.05);The bcl-2/Bax in 60? mol/L group was decreased to the blank control group(P<0.05),in 80? mol/L,100? mol/L group was significantly decreased to the blank control group(P<0.01);The Bax protein in 60?mol/L group was increased to the blank control gr oup(P<0.05)Retreated in 48h,the trophoblastic cell proliferation in 40?mol/L,60? mol/L was decreased compared to the control blank group(P<0.05);Th e early apoptosis rate in group 40?mol/L,60? mol/L,80? mol/L,100? mol/L was increased significantly than the blank control group(P<0.01);Induced in 48h,the trophoblastic cell Proliferation in 80?mol/L,100?mol/L was decreased compared to the control blank group(P<0.05);60? mol/L was significantly decreased than the control blank group(P<0.01);Retreated in 24h,the trophoblastic cell Proliferation in 100 mu mol/L group was significantly decreased than the control blank group(P<0.01);Retreated in 48h,the trophoblastic cell Proliferation in 80? mol/L g roup,100 ? mol/L group was significantly decreased than the control blan k group(P<0.01);The early apoptosis rate in group 80 ? mol/L,100?mol/L was increased significantly than the blank control group(P<0.01);Experiment 3:The trophoblastic cell proliferation in model control group decreased compared to the control blank group(P<0.01);and the ea rly apoptosis rate of model group was increased compared to the control b lank group(P<0.05)for 24h;The early apoptosis rate of model group was significantly increased compared to the control blank group for 48h(P<0.01);The Caspase-3 protein in the 10 group was decreased than the model control group(P<0.05).According to the results of stepwise regression,canonical correlation regression and the optimal analysis and the weight of each index weight,calculate the optimal proportion of active ingredi ents of the modified STW:Semen Cuscutae total flavonoids:Rumulus Taxill i total flavonoids:Rumulus Taxilli total Polysaccharides:Radix Dispaci Asperoidis total saponins = 0.9:0.00342:0.09037:0.0795(mg/ml).Experiment 4:In the optimal proportion group of the active ingredients of the modified STW compared to the model control group,the proliferation was significantly increased(P<0.01)in 24h;The early apoptosis rate was significantly decreased in 24h and 48h(P<0.01),The expression of Caspase-3 protein and mRNA,Bax protein and mRNA was significantly decreased in 24h(P<0.01);The expression of Caspase-9 protein was decreased in 24h(P<0.05);The level of ?-HCG in cell supernatants was no significant difference to the control model group(P>0.05);In the optimal group 10 compared to the model control group,the Proliferation of was increased(P<0.05)in 24h;The early apoptosis rate was significantly decreased in 24h(P<0.01);The expression of Caspase-3 protein and mRNA,Bax protein and mRNA was significantly decreased in 24h(P<0.01);The expression of Caspase-9 protein was decreased in 24h(P<0.05);The level of ?-HCG in cell supernatants was significant increased(P<0.01)in 48h.In the Semen cuscutae total flaconoids group compared to the model control group,the early apoptosis rate was decreased(P<0.05)in 24h;The expression of Caspase-3 protein significantly decreased in 24h(P<0.01);The expression of Bax protein and mRNA was significantly decreased in 24h(P<0.01);In the Hyperoside group compared to the model control group,the early apoptosis rate was significantly decreased in 24h(P<0.01);The expression of Caspase-3 protein was decreased in 24h(P<0.01);The expression of Caspase-9 protein was decreased in 24h(P<0.05);In the Asperosaponin VI group compared to the model control group,the early apoptosis rate was significantly decreased(P<0.01)in 24h;The expression of Caspase-3 protein was decreased in 24h(P<0.05),and significantly decreased in 48h(P<0.01);The expression of Caspase-9 protein was decreased in 24h(P<0.05);The expression of Bax protein was significantly decreased in 24h(P<0.01);Conclusion1.The Primary cultrured and degeneration conditions of trophoblastic for the further experiments in vitro was improvement.And the cells was identified to trophoblastic cells.2.The trophoblastic damaged model was established by RU486.The trop--hoblastic cells was inhibited of proliferation and induced of apoptosis by mifepristone in vitro,and the damage was continued retreated in 24h of 60?mol/L.3.Analysis and conclude trough the damage trophoblastic cells model,the optimal proportion of active ingredients of the modified STW is:Semen cuscutae total flaconoids:Rumulus Taxilli total flavonoids:Rumulu s Taxilli total polysaccharides:Radix Dispaci Asperoidis total saponins=0.9:0.00342:0.09037:0.0795(mg/ml).The optimal proportion of active ingr--edients of the modified STW can promote the damaged trophoblastic cells'proliferation,inhibit of the apoptosis though the inhibition of the exp ression of Caspase-3,Caspase-9 and Bax. |
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